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CN-116731985-B - Cytochrome P450 monooxygenase P450PL2 mutant and application thereof

CN116731985BCN 116731985 BCN116731985 BCN 116731985BCN-116731985-B

Abstract

The application discloses a cytochrome P450 monooxygenase P450PL2 mutant and application thereof in the technical field of biology. The P450PL2 enzyme from the detergent-carrying bacillus parvus DS-1 is subjected to multi-round directional transformation by the technical means of iterative saturation mutation, so that the P450PL2 mutant with improved asymmetric hydroxylation activity is obtained, and the corresponding amino acid sequence is shown as SEQ ID No. 3. The P450PL2 mutant can be applied to catalyzing asymmetric hydroxylation of aryl ketone compounds to synthesize S-configuration chiral alpha-hydroxy ketone compounds. The reaction has strict regioselectivity and stereoselectivity, and has simple and efficient operation process.

Inventors

  • CHEN YONGZHENG
  • WANG JING
  • Agui Iqbal Isa

Assignees

  • 中国科学院新疆理化技术研究所
  • 遵义医科大学

Dates

Publication Date
20260508
Application Date
20221104

Claims (6)

  1. 1. A cytochrome P450 monooxygenase P450PL2 mutant is characterized in that the amino acid sequence of the P450PL2 mutant is shown as SEQ ID NO. 3.
  2. 2. The P450PL2 mutant coding gene of claim 1, wherein the nucleic acid sequence is shown in SEQ ID No. 4.
  3. 3. A vector comprising a gene encoding the P450PL2 mutant of claim 2.
  4. 4. A genetically engineered bacterium comprising the vector of claim 3.
  5. 5. The cytochrome P450 monooxygenase P450PL2 mutant of claim 1, wherein the P450PL2 mutant is applied to catalyzing asymmetric hydroxylation reaction of aryl ketone compounds, wherein the aryl ketone compounds are propiophenone and 4-fluorophenylacetone.
  6. 6. The cytochrome P450 monooxygenase P450PL2 mutant of claim 5, wherein the asymmetric hydroxylation is carbonyl alpha asymmetric hydroxylation.

Description

Cytochrome P450 monooxygenase P450PL2 mutant and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a cytochrome P450 monooxygenase P450PL2 mutant and application thereof. Background Cytochrome P450 monooxygenases (CYPs) are a class of the heme-thiolate (heme-thiolate) protein superfamily, whose reduced form, when combined with CO, forms complexes which exhibit characteristic absorption peaks at 450 nm. As one of the largest superfamily of proteins, genes encoding P450 enzymes have been found in animals, plants, fungi, bacteria and protists. Among them, P450 enzymes are mainly involved in degradation of heterologous substances, drug metabolism, and synthesis of endogenous compounds in mammals, while they are used for biosynthesis of secondary metabolites and the like in plants and microorganisms. The P450 enzyme can play a role in biocatalysis on substrate molecules with various structure types under mild conditions, and the reaction types include hydroxylation reaction, epoxidation reaction, dealkylation reaction and the like. At present, with the development of technologies such as directed evolution and the like, more and more P450 enzymes are applied to fields such as drug synthesis and the like as important biocatalysts. Chiral alpha-hydroxy ketone is a ketone structural unit containing chiral hydroxy and widely exists in natural product molecules and drug molecules. In addition, chiral alpha-hydroxyketone can be used as an important intermediate to synthesize various drug molecules or active compounds, such as antidepressant (S) -bupropion, antifungal drug SCH 42427, alpha 4 beta 2-nAChR antagonist for smoking cessation treatment, hypoxia inducible factor-1 alpha inhibitor for resisting tumor, and the like. The asymmetric hydroxylation reaction of the alpha position of the carbonyl catalyzed by the P450 enzyme is an important green alternative scheme for chemically synthesizing chiral alpha-hydroxy ketone, and can avoid the use of expensive heavy metal catalysts, harsh reaction conditions and a large amount of organic solvents. At present, only a few research teams report asymmetric hydroxylation reactions of carbonyl alpha position catalyzed by P450 enzyme, such as P450BM3 mutant (Nature Chemistry 2011,3:738;Journal of Organic Chemistry 2015,80:950) and P450 154C2 (Biochemical and Biophysical Research Communications 2020, 522:355), but the problems of low regioselectivity, narrow substrate spectrum, low catalytic efficiency and the like exist. Disclosure of Invention In order to solve the technical problems, the invention provides a cytochrome P450 monooxygenase P450PL2 mutant and application thereof. The P450PL2 enzyme derived from the detergent-carrying parvobacterium DS-1 is subjected to multi-round directional transformation by a technical means of iterative saturation mutation, and the P450PL2 mutant V99L/L109Y/A124W/G134T/T179P/M182L/T259G/P299S/L303L with improved asymmetric hydroxylation activity is obtained. The P450PL2 mutant can be applied to catalyzing asymmetric hydroxylation of aryl ketone compounds to synthesize S-configuration chiral alpha-hydroxyketone compounds, has strict regioselectivity and stereoselectivity, and has simple and efficient operation process. In order to achieve the above purpose, the present invention provides the following technical solutions: a cytochrome P450 monooxygenase P450PL2 mutant has an amino acid sequence shown in SEQ ID NO. 3. Further, the mutant is a multiple site mutation comprising V99L, L109Y, A124W, G134T, T179P, M L, T259G, P299S and L303L. It is a second object of the present invention to provide a vector comprising a gene encoding the above P450PL2 mutant. The third object of the present invention is to provide a genetically engineered bacterium comprising the vector as described above. The fourth object of the invention is to provide the application of the P450PL2 mutant, the carrier or the genetic engineering bacteria in catalyzing the asymmetric hydroxylation reaction of aryl ketone compounds. Further, the asymmetric hydroxylation is carbonyl alpha asymmetric hydroxylation. Further, the aryl ketone compound has the following chemical formula: Wherein Ar represents an optionally substituted or unsubstituted aryl group, and R represents a hydrogen atom, a halogen atom, a nitro group, a nitrile group, a methyl group or a methoxy group. The working principle and the beneficial effects of the invention are that the P450PL2 mutant is named as P450PL 2-2D 11, the amino acid sequence of the P450PL2 enzyme is shown as SEQ ID NO.1, and all amino acid mutations in the 99 th, 109 th, 124 th, 134 th, 179 th, 182 th, 259 th, 299 th and 303 th positions of the amino acid sequence of the P450PL2 enzyme are shown as SEQ ID NO. 3. ① Compared with cytochrome P450 monooxygenase P450PL2 parent enzyme, the P450PL2 mutant provided by the invention has remarkably improved catalytic activity, can